Anti-nuclear antibodies (ANA) are a hallmark of systemic autoimmunity and of lupus erythematosus (SLE) in particular. Due to their prevalence in disease and specificity for ubiquitous self-antigens that normally induce self-tolerance, they have been the subjects of extensive investigation into mechanisms that lead to breaches in B cell self-tolerance during autoimmunity. Defining the origin of such antibodies, however, has proved to be problematic. This is because of germline sequence polymorphisms in Ig V region genes and because of extensive somatic diversity generated by processes that assemble V region genes during B lymphopoiesis in the bone marrow and that mutate them in the periphery during immune responses. To determine the origin of ANA, we developed a unique mouse model of spontaneous SLE in which V region gene expression by autoreactive B cells could be clearly defined and in which all somatic mutations could be unequivocally identified. In B6.Nba2 Tdt-/- Igh Igk mice, the absence of terminal deoxynucleotidyl transferase (Tdt) enabled us to identify every somatic mutation, even in CDR3, where untemplated addition of nucleotides during V gene assembly would otherwise obscure them. By reverting somatic mutations to germline sequence in spontaneous ANA-producing clones, we found in preliminary studies that almost all of the ANA originated from nonautoreactive antecedent B cells that acquired their autoreactivity via the process of somatic hypermutation (SHM). This finding leads us to hypothesize that activation-induced cytidine deaminase (AID) plays a paramount role in generating ANA of systemic autoimmune disease. In this application, we propose to test this mutation-founder hypothesis and its implications. We will use AID-deficient mice to determine if it applies to systemic autoimmunity in other spontaneous models of SLE and to specific types of ANA. We will determine if the requirement for T cell help in SLE, reported by numerous investigators, is explained solely by its role in generating the mutant autoreactive B cells or whether T cell help is also required after the mutant clones arise. Germline revertant nonautoreactive antibodies will be used as probes to define natural immunogens that recruited precursors to the ANA-producing mutant clones. We will develop a new model of tolerance in germline ANA-specific B cells and will determine whether mutant ANA generated by AID promote the escape of germline ANA-producing B cells by obscuring or removing nuclear antigens that would otherwise render the B cells tolerant. Our preliminary studies provide us with unique tools and information to address these issues. Results of this project will provide basic information about the natural history of autoreactive B cells, which is essential if we are to understand, control and prevent systemic autoimmune disease.